The first outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 were detected in France in 2010. P. syringae pv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine. P. syringae pv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandemrepeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains of P. syringae pv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains of P. syringae pv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes of P. syringae pv. actinidiae biovar 3 ICMP 18744 and of P. syringae pv. actinidifoliorum ICMP 18807. MLVA enabled the structuring of P. syringae pv. actinidiae biovar 3 and P. syringae pv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct from P. syringae pv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed for P. syringae pv. actinidifoliorum. We developed an MLVA scheme to explore the diversity within P. syringae pv. actinidiae biovar 3 and to trace the dispersal routes of epidemic P. syringae pv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes of P. syringae pv. actinidiae at a global level.A gricultural systems are continuously afflicted by emerging infectious diseases (1), which can have significant agronomic and economic consequences. A thorough knowledge of the causal agent (propagation and contamination pathways, suitable environmental conditions, host range, and pathogenicity) is essential for determining and implementing efficient disease-management measures. Pathogen genotyping yields precious information for understanding the diversity and population structure of the bacterial organisms responsible for outbreaks. It enables hypotheses about the dispersion routes of bacterial populations or clonal lineages involved in epidemics. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) (2) is a powerful and portable genotyping method. It has been demonstrated that MLVA has a higher sensitivity and resolution than any other genotyping methods, such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence type (MLST), applied for an in-depth study of bacteria populations or epidemic outbreaks (3, 4). The aim of MLVA is to use PCR to target the tandem repeats with a motif of more than five nucleotides and to analyze the variability of their pattern in order to discriminate isolates. Generally, VNTR loci evolve according to the stepwise mutation model (SMM) by gain or l...